The identification and analysis of clock genes in the fruit fly, Drosophila melanogaster, revealed that the circadian clock is based on autoregulatory feedback loops in gene expression. Similar feedback loops serve to keep circadian time in essentially all eukaryotes, and the genes that mediate feedback loop function are well conserved from insects to mammals. A key function of these feedback loops is that they drive circadian rhythms in transcription that are required to keep circadian time and drive overt rhythms in physiology, metabolism and behavior. Consequently, these clocks are of great clinical importance because their dysfunction can lead to sleep problems, metabolic disorders, and even cancer. Despite the importance of rhythmic transcription for circadian clock function, the mechanisms that regulate rhythms in transcription are not well understood. Feedback loop function is initiated when the bHLH-PAS proteins CLOCK (CLK) and CYCLE (CYC) form heterodimers and bind E-box regulatory elements to activate period, (per) and timeless (tim) transcription. Rising levels of PER and TIM proteins form complexes with DOUBLETIME (DBT) kinase, move into the nucleus, and bind CLK-CYC to inhibit transcription. Degradation of PER and TIM then releases repression, and the next cycle of transcription can begin. Even though transcriptional repression by PER-TIM- DBT controls rhythmic transcription, how these complexes repress transcription is not known. PER-TIM-DBT complexes bind to and remove CLK-CYC from E-boxes, thereby promoting transcriptional repression. PER- CLK binding is not sufficient to remove CLK-CYC from E-boxes, but PER-TIM-DBT dependent phosphorylation of CLK coincides with CLK-CYC release from E-boxes, suggesting that CLK phosphorylation represses transcription. Although DBT and PER are required for CLK phosphorylation, DBT catalytic activity is not necessary, indicating that other kinases directly phosphorylate CLK. In Specific Aim 1 we will identify and characterize kinases that directly phosphorylate CLK, define their target sites, and determine the function of these phosphorylation sites. When transcriptional repression is released by the degradation of PER and TIM, hyperphosphorylated CLK is replaced by transcriptionally competent hypophosphorylated CLK through an undefined mechanism. Overall CLK levels do not vary substantially over a circadian cycle, which suggests that hyperphosphorylated CLK is directly converted to hypophosphorylated CLK via dephosphorylation. In support of this possibility, CLK can be dephosphorylated by protein phosphatase 2a (PP2a) in cultured Schneider 2 (S2) cells. In Specific Aim 2 we will identify and characterize phosphatases that dephosphorylate CLK. This work will reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. PUBLIC HEALTH RELEVANCE: Essentially all organisms use circadian clocks to control daily rhythms in physiology, metabolism and behavior. These clocks are of great clinical importance since their dysfunction can lead to sleep problems, metabolic disorders, and even cancer. An understanding of the mechanisms that control clock function will lead to the development of treatments for clock disorders.